Cloning of GITF tilapia cathepsin B gene and expression analysis after infected by Streptococcus agalactiae

【Objective】 To explore expression difference of cathepsin B gene（CtsB）in GITF tilapias before and after infected with Streptococcus agalactiae，so as to provide scientific basis for studying immune response mechanism of tilapia resistant to S.agalactiae and obtaining new disease-resistant GIFT tilapia varieties of by SNP marker or genome editing technique.【Method】 Full-length cDNA sequences of CtsB gene in tilapia were cloned through RACE.Bioinformatics and phylogenetic analysis were conducted through online software such as SMART，Splign，MEGA v6.0，BoxShade and MegAlign.Tissue expression profile of CtsB gene in GIFT tilapia detected by quantitative real-time PCR（qRT-PCR）and change of CtsB gene expression after infected by S.agalactiae.【Result】Full-length cDNA sequences of CtsB gene was 1746 bp，containing a 161 bp 5'untranslated region（5'-UTR），a 592 bp 3'UTR，an open reading frame（ORF）of 993 bp，which encoded a total of 331 amino acid residues.Phylogenetic tree based on CtsB amino acid sequence similarity showed that GIFT tilapia was closely related to Oreochromis aureus，but was far from Cyprinus carpio，Scophthalmus maximus，Paralichthys olivaceus，Cynoglossus semilaevis and other species.The gene was expressed in liver，spleen，gill，kidney，brain，intestine，skin，and muscle of GIFT tilapia，and the highest expression was observed in the gills，which was 2.0-3.0 times higher than that in other organ tissues.After disease-resistant families and susceptible families of GIFT tilapia infected with S.agalactiae，relative expression of CtsB gene in spleen and kidney was up-regulated（P< 0.05），and it peaked after 50 h of infection.Fold of up-regulated expression of disease-resistant families was higher than that of susceptible families.【Conclusion】 CtsB gene is highly conservative in evolution of fish，but expression pattern varies significantly in species.CtsB gene is an important immune factor of GIFT tilapia and it bears important role of immunization against S.agalactiae，so it can be developed and utilized as SNP molecular marker of breeding disease-resistant tilapia.These results suggest that CtsB plays an important role in the immune response to S.agalactiae infection in GIFT tilapia.